Abstract
Protein methylation is one of the most common post-translational modifications observed in basic amino acid residues, including lysine, arginine, and histidine. Histidine methylation occurs on the distal or proximal nitrogen atom of its imidazole ring, producing two isomers: Nτ-methylhistidine or Nπ-methylhistidine. However, the biological significance of protein histidine methylation remains largely unclear owing in part to the very limited knowledge about its contributing enzymes. Here, we identified mammalian seven-β-strand methyltransferase METTL9 as a histidine Nπ-methyltransferase by siRNA screening coupled with methylhistidine analysis using LC–tandem MS. We demonstrated that METTL9 catalyzes Nπ-methylhistidine formation in the proinflammatory protein S100A9, but not that of myosin light chain kinase MYLK2, in vivo and in vitro. METTL9 does not affect the heterodimer formation of S100A9 and S100A8, although Nπ-methylation of S100A9 at His-107 overlaps with a zinc-binding site, attenuating its affinity for zinc. Given that S100A9 exerts an antimicrobial activity, probably by chelation of zinc essential for the growth of bacteria and fungi, METTL9-mediated S100A9 methylation might be involved in the innate immune response to bacterial and fungal infection. Thus, our findings suggest a functional consequence for protein histidine Nπ-methylation and may add a new layer of complexity to the regulatory mechanisms of post-translational methylation.
Highlights
In eukaryotic cells, one of the most common posttranslational modifications (PTMs) is protein methylation, that is, the transfer of a methyl group from SAM to lysine and arginine residues in the target protein [1,2,3,4]
A protein of interest is ectopically expressed as Flagtagged forms in human embryonic kidney 293T (HEK293T) cells and immunopurified with Flag affinity resin, followed by acid hydrolysis and subsequent analysis of the resulting amino acids by LC coupled to tandem MS (LC–MS/ MS)
Similar results were observed in Flag-tagged mouse S100A9 (Flag-S100A9) protein, but it should be noted that the signal intensity of Nπ-methylhistidine was increased
Summary
One of the most common posttranslational modifications (PTMs) is protein methylation, that is, the transfer of a methyl group from SAM to lysine and arginine residues in the target protein [1,2,3,4]. We established a new method that allows discrimination of the methylhistidine isomers of cellular proteins by using LC–MS/MS and combined it with an unbiased siRNA screen to identify molecules required for histidine Nπ-methylation of S100A9. A protein of interest is ectopically expressed as Flagtagged forms in human embryonic kidney 293T (HEK293T) cells and immunopurified with Flag affinity resin, followed by acid hydrolysis and subsequent analysis of the resulting amino acids by LC coupled to tandem MS (LC–MS/ MS).
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